Studies in this laboratory on the infection of Bacillus subtilis with phage SP82 and by other investigators on the related phage SP01 have established that the subunit structure of RNA polymerase is altered following infection (by loss of the sigma subunit and addition of three small peptides) and that the alteration causes a change in the transcriptional specificity of the enzyme. Evidence from this laboratory indicates that the phage-specified small peptides direct the enzyme to new promoters. It is proposed that this be tested by: 1) isolating and sequencing the SP82 promoters recognized by polymerases from uninfected and infected B. subtilis and 2) by studying the complexing of the different polymerases with promoter-bearing fragments of SP82 DNA. The promoter-bearing regions of SP82 DNA have been identified by hybridizing RNAs synthesized in vivo and in vitro to fragments of SP82 DNA produced by digestion with restriction endonucleases and by isolating DNA fragments which bind with different polymerases under conditions which permit initiation of RNA synthesis. Such fragments will be sequenced by the Maxam and Gilbert method. Initiation sites will be identified by sequencing short RNA chains synthesized in vitro. The sequencing data should yield a definitive answer as to whether the phage-induced peptides specify a new promoter regions. A second goal of the proposed investigation is to study the interaction of modified and unmodified polymerases with promoters on different DNA fragments. Filtration through nitrocellulose membranes and electrophoresis on agarose gels will be used to test the effect of ionic strength, temperature, nucleotide concentration, glycerol and the addition of enzyme subunits on the formation and stability of complexes. These investigations should provide information on factors influencing the selection of transcription sites by the different polymerases.